FR2901133A1 - Use of epidermal glycolysis-inducing compound in a composition, with exclusion of insulin growth factor type-1, as agent e.g. to reduce/delay the aging signs of skin and/or its annexes and to prevent/reduce e.g. wrinkles - Google Patents

Abstract

Use of an epidermal glycolysis-inducing compound in a composition containing a medium, with exclusion of insulin growth factor-type-1 (IGF-1), as an agent for reducing and/or delaying the aging signs of the skin and/or its annexes. An independent claim is included for a method of identifying the glycolysis inducing compound comprising preparing a keratinocyte culture, adding the compound to be tested in the cell culture medium, comparing the glycolysis levels or the GLUT-1 expression level in the keratinocytes in the presence of the compound to be tested with the glycolysis rate or the expression level in a sample without cultivated compound to be tested, and selecting the compound, which increases the glycolysis rate for at least 30% or increases the GLUT-1 expression for at least 25%. ACTIVITY : Nootropic; Dermatological; Metabolic. MECHANISM OF ACTION : Glucose transporters activator.

Description

Use of glycolysis modulators as anti-aging agents

  The invention relates to the use in a composition containing a physiologically acceptable medium, of at least one epidermal glycolysis inducing compound, excluding IGF-1 (Insulin Growth Factor type 1) as an agent for reduce and / or delay the signs of aging of the skin and / or its appendages.

  The present invention relates to the field of aging and the signs associated with it, on the skin and its appendages. It relates in particular to the modulation of the equilibrium between the proliferation and the differentiation of the epidermal cells and the improvement of the signs related to the phenomenon of thinning of the epidermis due to a decrease in the number of keratinocytes in the proliferation phase. Women, or even men, tend to want to look young as long as possible and therefore seek to fade the marks of aging skin, which result in particular in the form of wrinkles and fine lines, a thinning of the epidermis and / or a soft and withered skin appearance. In this regard, advertising and fashion mention products intended to keep as long as possible a glowing skin without wrinkles, marks of a young skin, especially as the physical aspect acts on the psyche and / or on morale. The skin consists of two compartments, one superficial, the epidermis, and one deeper, the dermis, which interact. The natural human epidermis is composed mainly of three types of cells, which are the keratinocytes, a very large majority, melanocytes and Langerhans cells. Each of these cell types contributes, by its own functions, to the essential role played in the body by the skin, in particular the role of protecting the body from external aggressions called "barrier function".

  The epidermis is conventionally divided into a basal layer of keratinocytes constituting the germinal layer of the epidermis, a so-called spinous layer consisting of several layers of polyhedral cells arranged on the germinal layers, one to three so-called granular layers consisting of flattened cells containing distinct cytoplasmic inclusions, keratohyalin grains and finally the stratum corneum, consisting of a set of layers of end-stage keratinocytes called corneocytes. Corneocytes are anucleate cells consisting primarily of a fibrous material containing cytokeratins, surrounded by a horny envelope. The dermis provides the epidermis with a solid support. It is also its nurturing element. It consists mainly of fibroblasts and an extracellular matrix mainly composed of collagen, elastin and a substance called the fundamental substance. These components are synthesized by fibroblasts. There are also leucocytes, mast cells or tissue macrophages. Finally, the dermis is crossed by blood vessels and nerve fibers.

  The cohesion between the epidermis and the dermis is ensured by the dermal-epidermal junction. 2 There is continuously in the epidermis a production of new keratinocytes to compensate for the continuous loss of epidermal cells in the stratum corneum. However, during aging, it is possible to physiologically observe a decrease in the number of cells in the proliferation phase, and consequently a decrease in the living epidermal layers. By limiting and / or delaying the passage of the cells in the differentiation phase, the pool of young cells is maintained. It is therefore important to preserve this pool of proliferative cells, avoiding or delaying their differentiation, in order to help delay the appearance of signs of aging. Retinoids, and especially retinol, are thus conventionally used to combat the signs of aging and promote epidermal renewal. However, there is still a need to find new agents useful in anti-aging.

  Therefore, the subject of the present invention is the use, in a composition containing a physiologically acceptable medium, of at least one epidermal glycolysis inducing compound, as an agent for reducing and / or delaying the signs of aging of the skin and / or or its appendices, including hair. Indeed, unexpectedly, it has been found within the scope of the invention that these compounds can be used as an agent for limiting the differentiation of epidermal cells. A basal pool of keratinocytes at the stage of proliferation is thus maintained.

  Preferably, said compound is not IGF-1 (insulin growth factor of type 1: insulin growth factor), or IGF-1 is not used as sole inducer of epidermal glycolysis. Advantageously, the glycolysis inducing compound is an activator of glucose transporter peptides (GLUT).

  Glucose transport in most mammalian cells is regulated by a family of membrane proteins called glucose transporter, also referred to as GLUT. At least 13 proteins are encoded by a family of related genes, these proteins having different transmembrane domains. The expression of the different isoforms of GLUT varies according to the tissue and the hormonal and environmental conditions.

  Keratinocytes express at least 4 types of glucose transporters, GLUT-1, 2, 3 and 5.

  Shen et al (J. Invest Dermatol., 2000) have described variations in transporter levels in murine keratinocytes upon stimulation with insulin or IGF-1. During differentiation, they observe an increase in the expression of GLUT-3 and a decrease in that of GLUT-1, 2 and 5. They indicate that the stimulation of keratinocytes being differentiated by insulin or IGF-1, has no effect on the expression of these GLUTs. In contrast, insulin activates the translocation (from the cytoplasm to the membrane fraction) of GLUT-1 and 5, and IGF-1 activates the translocation of GLUT-2 and 3 during the proliferation phase. The authors conclude that it would be interesting to compare the variations between normal skin and the skin of diabetic subjects. US 2003/0187036 proposes the use of biguanide derivatives to promote the healing of skin lesions such as ulcers occurring in diabetics, or other injuries. US 2005/0037440 relates to the identification of longevity factors in mammals, and to the role of the cJUN factor and the modulation of the JNK signal. He suggests using agents that decrease the activity of an indicator such as DAF-14, superoxide dismutase, GLUT-1 or GLUT-4 to fight against signs of aging caused mainly by oxidative stress. DE 10259966 discloses cosmetic or pharmaceutical compositions containing amino acids of the mycosporin type and improving the uptake of oxygen; these compounds induce in particular a reduction in the expression of GLUT-1. The compositions may in particular be applied for the treatment of aging of the skin.

  However, to the knowledge of the applicant, it has never been proposed to use glycolysis-inducing compounds or compounds capable of stimulating GLUT glucose transporter systems, in order to combat the signs of aging of the skin. or its annexes and to promote the renewal of epidermal cells.

  By activator compound of the glucose transporter peptides is meant both compounds increasing the amount of GLUT and agents increasing their intrinsic activity. The agents increasing the amount of GLUT are in particular compounds stimulating its synthesis, in particular by stimulating the expression or the transcription of the corresponding gene. The agents that increase the intrinsic activity of GLUT include agents that promote translocation of the protein into the cell membrane or glucose uptake.

  By glycolysis inducing compound is meant compounds increasing the reaction by which glucose is metabolized in the cells. The glycolysis comprises several steps: a first reaction chain results in the formation of fructosel, 6 di-phosphate, which is then degraded to pyruvate, with the production of ATP. Under anaerobic conditions, LDH catalyzes the conversion of pyruvate to lactate. The inventors have in fact demonstrated that anaerobic glycolysis is maximal in keratinocytes during the proliferation phase. On the other hand, differentiation is characterized by a significant decrease in glycolysis, without any qualitative modification of it. Preferably, GLUT transporter peptide activators are activators of GLUT-1, GLUT-2 and / or GLUT-3. It may be specific activator of one or more of these carriers, or nonspecific activators. Advantageously, the peptide activators useful according to the invention are activators of GLUT-1. It has been shown in the context of the invention that glucose transporters, in particular GLUT-1 and GLUT-2, vary according to the proliferation / differentiation state of keratinocytes and are involved in epidermal homeostasis. The modification of their activity can thus influence the control of the proliferation / differentiation balance during aging.

  The glycolysis-inducing and / or activating GLUT-activating compounds according to the invention may especially be chosen from hypoglycemic biguanides such as metformin, phenformin, buformin or proguanil; insulin; thiazolidone analogs, troglitazone; interleukins, in particular IL-3; protein phosphatase inhibitors such as vanadate, okadaic acid; dibutyryl cAMP and TGF-13. In general, compounds promoting the functioning of epidermal glycolysis, which are characterized by increased lactate production, are used.

  Other compounds that are useful according to the invention may be identified by the process comprising the following steps: a) culturing keratinocytes b) adding the test compound to the culture medium c) comparing the level of glycolysis in the keratinocytes presence of the test compound, with the rate of glycolysis in a control sample cultured without test compound d) selection of the compound which causes an increase of at least 30% in the glycolysis rate.

  Step a) may be carried out according to methods known to those skilled in the art, for example in autocrine medium or in complete medium in the presence of growth factors such as EGF, insulin, and pituitary gland extracts. In step b), the test compound is added at concentrations compatible with cell viability and generally from 10-2 to 10-9 M. It will be left in contact with the cell culture for a time sufficient to be metabolized by the cells. This time is usually greater than or equal to 12 hours. The level of glycolysis in step c) will be measured in keratinocytes in the proliferation phase and / or in keratinocytes in the differentiation phase; this rate will be compared with that observed in a control sample cultured in the absence of test compound, all the other culture conditions being identical. According to one embodiment of the invention, the variation in the level of glycolysis induced by the test compound is measured on the one hand in a population of keratinocytes, predominantly, even homogeneously, in the proliferation phase, on the other hand in a majority of keratinocyte population, even homogeneously, in the differentiation phase. This step of measuring the epidermal glycolysis may, if appropriate, be directly quantified either by measuring the PFK1 activity and / or by measuring glucose consumption and / or by measuring the level of GLUT expressed. by the cells and / or by the level of lactate produced during the overall reaction.

  The method according to the invention may thus comprise the following steps: a) Culture of keratinocytes and addition of the test compound in the culture medium b) Comparison of the level of glycolysis in the keratinocytes in proliferation and differentiation in the presence of the compound to test compared to a control sample grown without test compound. (c) This step of measuring epidermal glycolysis may, if appropriate, be directly quantified either by measuring PFK1 activity and / or by measuring glucose consumption and / or by measuring the level of GLUT expressed by the cells and / or by the level of lactate produced during the overall reaction. d) Selection phase of the compound which causes a significant activation of the parameters described in b) and / or c).

  The effects of the compounds identified by the method described above on the level of expression of GLUT-1 can be evaluated in a second step. This evaluation could be carried out according to the following steps: a) culturing keratinocytes b) adding the test compound in the culture medium c) comparing the level of expression of GLUT-1 in the keratinocytes in the presence of the test compound , with the level of expression in a control sample cultured without test compound d) selection of the compound which causes a significant increase, in particular of at least 25% of the expression of GLUT-1.

  Step a) may be carried out according to methods known to those skilled in the art, for example in autocrine medium or in complete medium in the presence of growth factors such as EGF, insulin, and pituitary gland extracts.

  In step b), the test compound is added at concentrations compatible with cell viability and generally from 10-2 to 10-9 M. It will be left in contact with the cell culture for a time sufficient to be metabolized by the cells. This time is usually greater than or equal to 12 hours. The level of expression in step c) will be measured in keratinocytes in the proliferation phase and / or in keratinocytes in the differentiation phase; this expression will be compared with that observed in a control sample cultured in the absence of test compound, all the other culture conditions being identical. This step of measuring the level of expression of GLUT-1 can, if appropriate, be directly quantified either by the quantification of the signal following the immunolabelings of the keratinocytes with a specific antibody for GLUT-1 or by the quantification by RT- PCR mRNAs encoding GLUT-1 and / or by measuring the expression of the protein by Western blot. The compounds that are useful according to the invention can thus be selected by a process comprising the following steps: a) Culture of keratinocytes and addition of the test compound in the culture medium b) Comparison of the level of expression of GLUT-1 in keratinocytes in proliferation and differentiation in the presence of the test compound compared to a control sample cultured without test compound. c) This step of measuring the expression of GLUT-1 can, if appropriate, be directly quantified either by the quantification of the signal following immunolabelings of keratinocytes with a specific antibody for GLUT-1 and / or by the quantification by RT-PCR of the mRNAs encoding GLUT-1 and / or by measuring the expression of the protein by Western blotting. d) Selection phase of the compound which causes a significant increase in the parameters described in b) and / or c).

  It is understood that the methods for selecting suitable compounds and their variants are also subject of the invention.

  Compounds regulating glucose metabolism by activating or increasing glucose transport in cells, more particularly in keratinocytes, can increase their proliferation and / or slow down their differentiation, thus keeping them in a proliferative state corresponding to young cells. This activation of the proliferation and / or this decrease in differentiation is objectified, in particular, by the increase in the incorporation of tritiated thymidine or by the decrease in the expression of certain markers of differentiation among which, keratin 1 and / or or keratin and / or transglutaminase K and / or loricrin and / or fillagrin. K10 (keratin 10) is a protein that serves as a marker of early differentiation. It is coexpressed with K1 as a heterodimeric protein. It is detected in all the keratinocytes of the suprabasal layers of the epidermis, ie from the beginning of the differentiation process. TGK (Transglutaminase K) is a marker of late differentiation. It is an enzyme involved in the crosslinking of the horny envelope of keratinocytes of the suprabasal upper layers.

  The compounds inducing glycolysis according to the invention will be particularly useful as an agent for combating the signs of chronobiological aging of the skin and its appendages.

  By skin, is meant in this text, the skin, mucous membranes and / or the scalp. By annexes of the skin is meant in particular the superficial body growths, that is to say, the hairs, the eyelashes, the hair and / or the nails. The compounds will be particularly suitable for combating the signs of chronobiological aging of the epidermis. During chronobiological aging, the thickness of the epidermis is reduced, cell divisions decreasing in number. By facilitating cell multiplication, especially epidermal cells, it will facilitate its regeneration and the skin will look younger.

  The compositions according to the invention are especially intended to prevent or reduce wrinkles and fine lines, and / or thinning of the skin and / or against soft and / or faded skin.

  According to another aspect of the invention, the glycolysis inducing compound and / or GLUT activator or the compositions containing it are used to combat the signs of aging of the integuments. The compound is thus used as an agent for reducing and / or preventing hair loss, and / or for preventing heterogeneity of the capillary diameter. Indeed, by promoting the hair follicle, the maintenance of keratinocytes proliferation phase, contributes to the formation of a hair shaft and its renewal. Said compounds or compositions are especially intended for the treatment or prevention of alopecia. During the course of alopecia, the aging of the whole epithelial structure of the hair follicle is observed, in particular with a decrease in the growth and diameter of the hair shaft that is directly related to a functional alteration of the epithelial cell renewal linked to the hair follicle. aging of the structure or indirectly to a lack of oxygenation or nutrition or to a degradation of the functionality of the connective structures of support of the organ.

  According to another aspect of the invention, the glycolysis inducing compound and / or GLUT activator is used as an agent for combating cutaneous dehydration and / or dryness of the epidermis. It is used as a moisturizing agent, in compositions intended to promote the synthesis of NMF (natural moisturizing factor) constituent element for the barrier function and hydration of the epidermis. This synthesis is favored through the production of lactate, which is a major constituent of the NMF.

  Skin dryness can be related to aging skin, and be one of the signs. But the compounds are also useful for controlling the dry conditions constitutive of the skin, which may exist physiologically in a subject regardless of age.

  According to another embodiment, the glycolysis inducing compound as defined above, is used for the preparation of a composition intended to treat disorders related to abnormal differentiation of the epidermis, such as hyperkeratosis.

  The compositions according to the invention may be cosmetic or pharmaceutical compositions, in particular dermatological compositions.

  By physiologically acceptable medium is meant a medium compatible with the skin, mucous membranes or integuments. It is in particular a cosmetically or pharmaceutically acceptable medium.

  The amount of glycolysis-inducing compound in the compositions is dependent on the desired effect and will be adapted by those skilled in the art to provide stimulation of proliferation or decrease in keratinocyte differentiation. For example, the amount of glycolysis inducing agent or glucose transport can vary from 0.0001% to 10% and preferably from 0.001 to 5% by weight relative to the total weight of the composition.

  Preferably the compositions are cosmetic compositions, that is to say intended to improve the appearance of the individual.

  For administration by the oral route, the composition of the invention may be in any suitable form, particularly in the form of an oral solution, a tablet, a capsule, a capsule or even a nutritional food or a nutritional supplement.

  The composition further comprises at least one suitable excipient suitable for oral administration.

  For administration by topical application to the skin, the hair and / or the mucous membranes, the composition according to the invention obviously comprises a cosmetically acceptable support, ie a support compatible with the skin, the mucous membranes, the nails, the hair and can be in all the galenical forms normally used for topical application, especially in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water or water-in-oil or multiple emulsion, an aqueous or oily gel, a liquid, pasty or solid anhydrous product, a suspension or a dispersion; for example an oil dispersion in an aqueous phase using spherules, these spherules may be polymeric nanoparticles such as nanospheres and nanocapsules or better lipid vesicles of ionic and / or nonionic type.

  This composition may be more or less fluid and have the appearance of a white or colored cream, an ointment, a milk, a lotion, a serum, a paste, a mousse . It can optionally be applied to the skin in the form of an aerosol. It can also be in solid form, and for example in the form of a stick. It can be used as a care product, as a cleaning product or as a makeup product.

  In known manner, the composition of the invention may contain the usual adjuvants in the cosmetic and dermatological fields, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers , filters, pigments, chelating agents, odor absorbers and dyestuffs. The amounts of these various adjuvants are those conventionally used in the fields under consideration, and for example from 0.01% to 20% of the total weight of the composition. These adjuvants, depending on their nature, may be introduced into the fatty phase, into the aqueous phase, into the lipid vesicles and / or into the nanoparticles. When the composition of the invention is an emulsion, the proportion of the fatty phase may from 5% to 80% by weight, and preferably from 5% to 50% of the total weight of the composition. The oils, emulsifiers and coemulsifiers used in the composition in emulsion form are chosen from those conventionally used in the field under consideration. The emulsifier and the coemulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% of the total weight of the composition.

  As oils that can be used in the invention, mention may be made of mineral oils, oils of vegetable origin (apricot oil, sunflower oil), oils of animal origin, synthetic oils, silicone oils and oils. fluorinated (perfluoropolyethers). It is also possible to use fatty alcohols (cetyl alcohol), fatty acids and waxes (beeswax) as fatty substances.

  As emulsifiers and coemulsifiers that can be used in the invention, mention may be made, for example, of fatty acid and polyethylene glycol esters such as PEG-40 stearate, PEG-100 stearate, fatty acid and polyol esters. such as glyceryl stearate and sorbitan tristearate.

  As hydrophilic gelling agents, mention may in particular be made of carboxyvinyl polymers (carbomer), acrylic copolymers such as copolymers of acrylates / alkylacrylates, polyacrylamides, polysaccharides, natural gums and clays, and, as lipophilic gelling agents, it is possible to mention mention modified clays such as bentones, metal salts of fatty acids, hydrophobic silica and polyethylenes.

  According to an advantageous embodiment, the compositions according to the invention will contain at least one other active agent chosen from UV-screening agents, moisturizing agents, depigmenting agents, anti-glycation agents, NO synthase inhibitors, synthesis of dermal or epidermal macromolecules and / or preventing their degradation, agents stimulating the proliferation of fibroblasts or keratinocytes, myorelaxing or dermo-decontracting agents, tensing agents, anti-pollution or anti-radical agents, soothing agents and the active ones on the energetic metabolism of the cells. The amount of these additional active ingredients may vary to a large extent and will for example be from 10-6 to 20% by weight, especially from 0.001 to 10% by weight relative to the total weight of the composition.

  The agents stimulating proliferation of fibroblasts that can be used in the composition according to the invention may for example be chosen from plant proteins or polypeptides, extracted especially from soybeans (for example a soybean extract marketed by LSN under the name Eleseryl SH-VEG). 8 or marketed by SILAB under the trade name Raffermine); and plant hormones such as giberrellins and cytokinins.

  The agents stimulating the proliferation of keratinocytes, usable in the composition according to the invention, include retinoids such as retinol and its esters, including retinyl palmitate; adenosine, cinnamic acid and its derivatives, lycopene and its derivatives; phloroglucinol; nut cake extracts sold by the company GATTEFOSSE; and extracts of Solanum tuberosum marketed by SEDERMA.

  The subject of the invention is also a cosmetic treatment method for preventing and / or reducing the signs associated with aging of the skin or its appendages, in which the skin, the mucous membranes and / or the scalp are applied to at least one skin. compound inducing glycolysis and / or GLUT activator as defined above. The compound will preferably be applied in a cosmetic composition, the application being daily, multi-daily or weekly. It can be continued for several days or weeks, for example for 1 to 2 months and then resume after an interruption, or be continuous.

  Other features and advantages of the invention will appear on reading the examples which follow. In these examples, reference is made to the following figures. FIG. 1: Immunolabelling of histological sections of human skin showing the differential expression of GLUT1 and GLUT-2 transporters in epidermal keratinocytes.

  Figure 2: Curve showing the effect of different concentrations of metformin on glucose transport in proliferating keratinocytes. 3: Activator effect of metformin on the expression of GLUT-1 in keratinocytes in the differentiation phase.

  Figure 4: Inhibitory effect of metformin on the expression of markers of epidermal differentiation. Results observed on monolayers of differentiating keratinocytes.

  Example 1 Evolution of glycolysis in keratinocytes. Human epidermal keratinocytes are isolated, according to the technique described by Wille et al. (1984), from normal skin samples from abdominoplasty. The keratinocytes are first cultured in primary culture in a serum-free complete medium (KBM base medium, Cambrex, supplemented with EGF, insulin, hydrocortisone, bovine pituitary gland extract and gentamycin). The proliferating keratinocytes from subconfluent cultures are trypsinized (trypsin / 1X EDTA, Invitrogen) and inoculated at 10 X 10 3 cells / cm 2 in the same complete medium. When the cells cover about 40% of the culture surface, the keratinocytes synthesize their own growth factors in sufficient quantity. The complete medium is then replaced by an autocrine medium containing only the basal medium, hydrocortisone and gentamycin. Subconfluent cultures of proliferating keratinocytes correspond to 24 hours of autocrine culture. The entry into confluence of the cells (3 days after the autocrine culture) induces the arrest of the cell proliferation and marks the initiation of the differentiation process (Poumay and Pittelkow, 1995, Poumay et al., 1999a). The post-confluent cultures of differentiating keratinocytes correspond to 6 days of autocrine culture (or 3 days of confluent culture).

  The rate of glycolysis is measured by the detritiation method of [2-3H] Glucose (glucose uptake) and [3-3H] Glucose (glycolytic flux).

  Measurement of the detritiation of f2-3HlGlucose (glucose uptake) and f3-3H1Glucose (glycolytic flux).

  Glucose uptake (glucose transport and phosphorylation) is evaluated by the detrimentation rate of [2-3H] glucose while gycolytic flux through PFK1 is estimated by the detrimentation rate of [3-3H] glucose. The keratinocytes cultured under autocrine conditions are incubated in the presence of fresh medium containing 6 mM glucose.

  After 2 hours, the radioactive tracer ([2-3H] glucose or [3-3H] glucose) is added to the culture medium (0.25 Ci / ml). After 30 to 60 minutes of incubation, the culture medium is recovered to measure the formation of 3H2O. These samples are first deproteinized on ice with perchloric acid (7% final). After neutralization with 3M / 3M KOH / KHCO3, the samples are centrifuged (1000 g, 10 min, 4 C). 3H2O is separated from radioactive glucose by column chromatography (AGX8 exchange resin in borate form). The results are expressed in nmol of glucose per hour and per μg of protein.

Determination of lactate.

  The lactate assay is carried out by measuring the formation of NADH (reading at 340 nm) by lactate dehydrogenase (LDH). The reaction is carried out in the presence of 25 to 100 μl of deproteinized and neutralized culture medium (see Measurement of [2-3H] Glucose and [3-3H] Glucose), hydrazine buffer pH 9.3 (hydrazine sulfate 0). , 4M, 1M glycine, 5mM EDTA), 2mM final NAD. After a first reading at 340 nm (E0), the reaction is initiated with 50 μg LDH. The second reading at 340 nm is made at the reaction plate (after about 30 minutes) (El). The result is expressed in nmole taking into account the molar extinction coefficient of NADH (6200).

  The following results are observed: Table 1 shows the glucose metabolism in proliferating and differentiating keratinocytes, in the presence or absence of growth factors (respectively autocrine culture and culture in complete medium). The values are expressed in nmoles / g protein / h and represent the SD average of at least 3 independent experiments.

  Glycolytic Flux Transport Lactose Glucose Production Autocr 0.65 0.07 0.99 0.19 1.62 0.20 prolif proliferating at Autocrine 0.20 0.05 0.21 0.05 0.43 0 , 05 Differentiation Complete in 1.17 0.11 1.52 0.21 3.04 0.33 Proliferation (Growth Factors) Complete in 0.53 0.13 0.43 0.03 0.75 0.05 Differentiation (growth factors) a) during the proliferation phases the glycolysis is maximal in keratinocytes. This glycolysis is anaerobic type with a production of 2 lactates for a glucose consumed. Whatever the experimental conditions, epidermal glycolysis is of the anaerobic type and leads to the production of two lactates per molecule of glucose. The values obtained are comparable to those observed in very high energy cells such as muscle cells in contraction: striated muscle cells or cardiac muscle cells. b) Differentiation induces a significant decrease in glycolysis.

  In conclusion, the phases of proliferation and differentiation of the cutaneous epithelium are correlated with glucose metabolism. During the transition to differentiation this glycolysis decreases.

  Example 2: Control of glycolysis in the epidermis: Expression of GLUT-1 and GLUT-2

  Glucose is transported by diffusion facilitated via transmembrane transporters, GLUTs (GLUT-1-12 and HIMT), the expression of which varies according to tissues and hormonal and environmental conditions. Keratinocytes express at least 4 types of glucose transporters (GLUT-1,2,3 and 5).

  Immunofluorescence detection of GLUT expression.

  Skin samples frozen in a coating medium and stored at -80 ° C are cut with a cryostat. The sections are fixed in acetone at 4 ° C. for 10 min and then dried in air. Non-specific sites are saturated by incubation in Ca / Mg-free PBS (ICN Biomedicals) supplemented with 0.1% triton-X-100 and 10% BSA (Bovine Albumin Fraction V, MP Biomedicals) for 1 hour at 37 minutes. C in humid chamber.

  The sections are then incubated in the presence of the specific primary antibody (anti-GLUT-1, rabbit, Santa Cruz, 1/100, anti-GLUT-2 Ab, rabbit, Santa Cruz, 1/100) diluted in PBS / BSA 1% / 0.01% Triton-X-100) for 1 hour at 37 ° C. in a humid chamber. After washes in PBS, the sections are incubated in the presence of the secondary anti-rabbit antibody Alexa Fluor 598 (Molecular Probe, 1/800, PBS / 1% BSA / 0.01% Triton-X-100) for 1 hour. at 37 C in a humid chamber. The nuclei are labeled with a 10 min incubation with Hoescht (1/1000, PBS / 1% BSA / 0.01% Triton-X-100). The sections are washed again in PBS, mounted and observed under a fluorescence microscope.

  Western blot analysis of the expression of GLUTs in cultured keratinocytes. The keratinocytes are cultured according to the protocol described in Examplel. The proliferating keratinocytes are removed at 1 day of autocrine culture. The differentiating keratinocytes are removed at 6 days of autocrine culture. For the detection of GLUT-1, the keratinocytes are lysed in triton buffer (20 mM Hepes, pH 7.6, 20 mM NaF, 30 mM KCl, 1 mM EDTA, protease inhibitors and phosphatases, 1% triton). For the detection of GLUT-2, the keratinocytes are lysed in SDS buffer (62.5 mM Tris, pH 7.5, 1% SDS). Proteins (15 g / lane) are separated by SDS-10 gel and transferred to PVDF membrane. Saturation of the sites for 1 hour in TBS / 0.1% Tween / 5% milk.

  The primary antibody is incubated for 1 h in the saturation buffer (Anti-Glucose Transporter-1, Human (rabbit) Calbiochem, ref 400060, 1/1000, Anti-Glucose Transporter-2, rat (rabbit) Calbiochem, ref 400061, 1/1000). The secondary antibody is incubated for 1 h (anti-rabbit / peroxidase antibody Sigma A-0545, 1/20000) in 0.1% TBS / Tween. Use of the ECL kit (Roche) for the revelation.

  The expression of GLUT-1 and GLUT-2 transporters observed on epidermal sections varies according to the epidermal localization: GLUT-1 is expressed mainly in proliferating cells - the basal layer - while GLUT-2 is expressed preferentially in differentiating cells.

  The preferential expression of GLUT-1 in the proliferation phase and GLUT-2 in the differentiation phase is also observed by the Western Blots technique or a band at 51 kDa corresponding to the labeling by the anti-GLUT-antibody is observed. 1 for cells in the proliferation phase. This band is not visible for differentiating cells. The results are shown in Figure 1 in the appendix. GLUT-1 is visualized in the basal layers of the epidermis and GLUT-2 in the differentiated layers up to the stratum corneum.

  Example 3 Effect of metformin on glucose transport, GLUT-1 expression and the differentiation process in keratinocytes. The keratinocytes, at day 1 of autocrine culture, are treated for 2.5 hours with increasing doses of metformin (0-10 mM). The radioactive tracer ([2-3H] Glucose, 0.25 Ci / ml) is added during the last 50 minutes of the incubation. After incubation, the culture media are first deproteinized on ice with perchloric acid (10% final). After neutralization with 3M / 3M KOH / KHCO3, the samples are centrifuged (1000 g, 10 min, 4 C). 3H2O is separated from radioactive glucose by column chromatography (AGX8 exchange resin in borate form). The results are expressed in nmol of glucose per hour and per μg of protein.

  The results are shown in Figure 2 in the appendix. Metformin increases, in a dose-dependent manner, the transport of glucose into keratinocytes. This increase is related to an increase in the expression of GLUT-1.

  Activating effect of metformin on the expression of GLUT-1 in keratinocytes. The keratinocytes are treated with either metformin (2 mM) or EGF (10 ng / ml) or insulin (5 g / ml) from day 3 of autocrine culture (95% confluence) and for 72 hours. (renewal of treatment after 48 hours of treatment). The RNAs are extracted using the High Pure RNA isolation kit (Roche) as specified by the supplier. RNA (1 g / condition) is retro-transcribed for 2 h at 37 C. The cDNA corresponding to RNAs encoding GLUT-1 is amplified by means of specific primers for 25 cycles. The quantitative PCRs are carried out with Platinium SYBR Green (Invitrogen) on a MyIQ thermal cycler (Bio-Rad) according to the indications of the supplier. The amplification of 36B4 is used as an internal control.

  An increase in the expression of GLUT-1 in keratinocytes is observed for metformin. The results are presented in Figure 3 in the appendix.

  Effect of glvcolvtic flow modification on keratinocyte differentiation. 18

  The keratinocytes are treated with 2 mM metformin from day 3 of autocrine culture (95% confluence) and for 72 hours (renewal of the treatment after 48 hours of treatment). The RNAs are extracted using the High Pure RNA isolation kit (Roche) as specified by the supplier. The RNA (1 g / condition) is retro-transcribed for 2 hours at 37 ° C. The cDNA corresponding to the RNAs encoding keratin 10, involucrine, loricrin and transglutaminase 1 are amplified by means of specific primers. for 25 cycles. The quantitative PCRs are carried out with Platinium SYBR Green (Invitrogen) on a MyIQ thermal cycler (Bio-Rad) according to the indications of the supplier. The amplification of 36B4 is used as an internal control.

  Metformin, which increases glucose transport, significantly inhibits differentiation processes. There is therefore a close correlation between glycolysis and proliferation / differentiation. The results are shown in Figure 4 in the appendix.

Claims (12)

claims   1- Use in a composition containing a physiologically acceptable medium, at least one epidermal glycolysis inducing compound, excluding IGF-1 (Insulin Growth Factor type 1) as an agent for reducing and / or delaying signs of aging of the skin and / or its appendages.   2- Use of at least one glycolysis inducing compound according to claim 1 as an agent for limiting the differentiation of epidermal cells.   3- Use according to at least one of claims 1 or 2, characterized in that the glycolysis inducing compound is an activator of glucose transporter peptides (GLUT)   4- Use according to claim 3, characterized in that at least one GLUT activator compound is selected from the group comprising agents stimulating the synthesis of GLUT and agents increasing its intrinsic activity. 20   5- Use of at least one glycolysis-inducing compound according to any one of claims 3 or 4, characterized in that it is an activator of GLUT-1, GLUT-2 and / or GLUT-3 .   Use of at least one glycolysis inducing compound according to any one of claims 1 to 5, characterized in that at least one compound is selected from metformin, phenformin, buformin, proguanil, insulin, protein phosphatase inhibitors such as vanadate, okadaic acid, dibutyryl cAMP and TGF-13. 30   7- Use of at least one glycolysis-inducing compound according to any one of claims 1 to 6, characterized in that this compound is identified by the process comprising the following steps: a) culturing of keratinocytes 35 b) addition c) Comparison of the level of glycolysis in the keratinocytes in the presence of the test compound with the rate of glycolysis in a control sample grown without test compound d) Selection of the compound which causes a test compound increase of at least 30% in the rate of glycolysis   8- Use of at least one glycolysis-inducing compound according to any one of claims 1 to 7, characterized in that this compound is identified by the process comprising the following steps: i) culturing keratinocytes ii) adding the compound to be tested in the culture medium iii) comparison of the level of expression of GLUT-1 in the keratinocytes in the presence of the test compound with the level of expression in a control sample cultured without test compound iv) selection of the compound increases the expression of GLUT-1 by at least 25%   9- Use of at least one glycolysis-inducing compound according to one of claims 1 to 8, characterized in that said compound is used as an agent for combating the signs of chronobiological aging of the epidermis.   10- Use according to claim 9 characterized in that the inducer of glycolysis is intended to prevent or reduce wrinkles and fine lines, and / or thinning of the skin and / or against soft skin and / or withered.   11-Use of at least one glycolysis inducing compound according to one of claims 1 to 8, characterized in that said compound is used as an agent for reducing and / or preventing hair loss, and / or for preventing heterogeneity of the capillary diameter. 30   12- Use of at least one glycolysis-inducing compound as defined in one of claims 1 to 8, in a composition containing a physiologically acceptable medium, characterized in that said compound is used as an agent for combating cutaneous dehydration and / or the dryness of the epidermis.